Colorectal carcinoma (CRC), including colon and rectal cancer, is a leading cause of cancer deaths in the Western world but reliable biomarkers useful for early diagnosis and targeted therapy of this disease are lacking. The tumor-associated carbohydrate antigens (TACAs) termed Tn (GalNAc1-Ser/Thr) and sialylTn (STn) (NeuAc2-6GalNAc1-Ser/Thr) often appear at an early stage of colon carcinogenesis, and are associated with poor prognosis and tumor metastasis. However, the genetic basis for Tn/STn antigen expression is unclear. We recently found that expression of the Tn/STn antigens can arise from loss-of-function of T-synthase, required for normal core 1 O-glycans biosynthesis, due to its incorrect folding as a result of compromised expression of its specific molecular chaperone Cosmc, which is encoded by an X-linked (Xq24) gene. We showed that in human tumors and tumor cell lines, acquired mutations in Cosmc, which include point mutations in the coding region, deletion of the gene, loss-of-heterozygosity, and hypermethylation of its promoter, can result in a dysfunctional Cosmc associated with loss of T-synthase activity and expression of Tn/STn. Our preliminary studies now show that gastrointestinal (GI) tract epithelial cell-specific loss of Cosmc in mice causes Tn/STn antigen expression in the small and large intestine. We hypothesize that Tn and STn tumor antigens in CRC are novel glycan biomarkers for human colon cancer and arise from alterations in Cosmc or altered expression of T-synthase and/or C3GnT. We will explore this hypothesis in 4 specific aims. Aim 1: define the expression of Tn/STn and other TACAs in human primary colon tumors; Aim 2: compare glycan and glycopeptide profiles of colorectal carcinoma cells to normal cells; Aim 3: define the molecular mechanism(s) for Tn/STn expression in human primary colon tumors by identifying the alterations in Cosmc, as well as analyze transcript levels for other glycomics-relevant genes; and Aim 4: develop well- defined monoclonal antibodies that exhibit both high specificity and affinity to Tn or STn antigens, which could be used in both diagnosis and treatment of CRC. This project will define the molecular basis for Tn and STn expression in CRC and their potential as novel glycan biomarkers for human colon cancer. PUBLIC HEALTH RELEVANCE: The studies proposed will identify for the first time the molecular basis for expression of the tumor antigens Tn and Sialyl Tn in glycoproteins of human colorectal carcinoma (CRC), a leading cause of cancer deaths in the Western world. The studies will provide new glycomic information about CRC as well as generate new monoclonal antibodies with high affinity and specificity for Tn and STn antigens, that could be used in both diagnosis and treatment of CRC.